Title: Role of micro-RNAs in T cells and other immune/ hematopoietic cells Abstract MicroRNAs (miRNAs) are short, ~21 nucleotide (nt) RNA molecules that modulate gene expression by influencing messenger RNA (mRNA) stability in a variety of eukaryotic organisms. The miRNA pathway is evolutionarily very highly conserved, and individual miRNAs have been demonstrated to play important roles in cell differentiation, proliferation, apoptosis, cell lineage specification and cancer. We have previously documented distinct miRNA expression patterns in selected classes of murine hematopoietic cells, among them naive T cells and their progeny Thl and Th2 cells. We have also shown that T cells lacking the RNase -like enzyme Dicer, which performs the last step of miRNA processing in the cytoplasm, exhibit a Th1 bias;and that conversely, T cells lacking the siRNA/ miRNA exonuclease mExo preferentially differentiate into Th2 cells. This proposal describes an extension of these studies, in which the functions of selected miRNas in the immune and hematopoietic systems will be systematically investigated, and the role of gene products produced by target mRNAs will be investigated. In Aim 1. the role of miR-146 and its target TRAF6 in T cell differentiation will be explored, focusing on our recent finding that the transcription factor NFAT, which influences almost every aspect of T cell function, is a TRAF6-interacting protein In Aim 2. the functions of miRNAs 146, 221/222 and 223 in T cell/ hematopoietic cell differentiation will be investigated, primarily through targeted disruption in ES cells and in mice. In Aim 3. new miRNAs with potential roles in Th1/Th2 differentiation and their possible target mRNAs will be identified through unbiased cloning, deep sequencing and transcriptional profiling by microarray analysis. Together these studies should greatly increase our understanding of the role of miRNAs in T cells and other cells of the immune and hematopoietic systems.